Luminometric single step urea assay using ATP-hydrolyzing urease.

An automatic enzyme kinetic luminometric method for determination of small quantities of urea in biological fluids and in microdialysates is presented. The method is based on the ATP-hydrolyzing urease reaction [urea amidohydrolase (ATP-hydrolyzing); EC 3.5.1.45], monitored by a luciferin-luciferase ATP reaction. The assay range is 100 pmol to 50 nmol with a detection limit of 5 micromol/L in the sample, compared with detection limits of 0.1 mmol/L in earlier spectrophotometric methods. To reduce the non-urea-dependent ATPase activity (v(blank)) and to increase the urea-dependent activity, 1,2-propanediol was included. Assay conditions were optimized by multivariate analysis. Recoveries of urea added to blood dialysate and plasma were 96-103%. No analytical interference of common metabolites, drugs, or other additives was observed. The total CVs (6 days and six concentrations, 1.2-21.8 mmol/L) were 3.6-8.5%. The results obtained with the present assay were highly correlated for dialysate (r = 0.979) and for plasma (r = 0.978) with those obtained by a spectrophotometric kit method with slopes of 1.02-1.03 and intercepts of 0.08-0.23 mmol/L.

Release of small amounts of free fatty acids from human adipocytes as determined by chemiluminescence.

A semiautomatic luminometric method for determination of small amounts of free fatty acids (FFA) released from human adipocytes in vitro is described. Bovine serum albumin (BSA) is used as acceptor of free fatty acids in the incubation medium of isolated fat cells. The assay involves pretreatment with the detergent sodium dodecyl sulfate (SDS) to liberate the free fatty acids from the bovine serum albumin before activation by acyl-CoA synthetase (ACS) (EC 6.2.1.3). This is followed by oxidation of the resulting thioesters by acyl-CoA oxidase (ACO). The H2O2 formed is subsequently measured in a horseradish peroxidase (HRP) (EC 1.11.1.7)-catalyzed luminol reaction. The assay is linear in the interval of 0.01-1 nmol in the cuvette corresponding to 2-200 microM in the sample, and 25 samples are automatically assayed in the luminometer within 75 min. FFA release could easily be studied in a small incubation volume (200 microliters) of very diluted (10(4) cells/ml) human adipocyte suspensions. Samples (25 microliters) containing 0.25% BSA from incubates of adipose tissue cells did not interfere with the standard curve. The analytical interference from different factors that could be used in studies of lipolysis was investigated. No interference was observed up to the following concentrations: 5 microM epinephrine, 5 microM norepinephrine, 80 microM isoproterenol, 1 mM insulin, 2.5 mM propranolol, 5 mM phentolamine, and 5 microM ascorbate. Results obtained with the present assay were highly correlated (r = 0.997) with those obtained by a 260-times less sensitive spectrophotometric kit method.(ABSTRACT TRUNCATED AT 250 WORDS)

Effects of insulin deprivation and replacement on in vivo subcutaneous adipose tissue substrate metabolism in humans.

The effects of insulin deprivation and replacement on adipose tissue metabolism were investigated in vivo with microdialysis in nine insulin-dependent diabetic patients with no residual insulin secretion. Dialysis probes, implanted in abdominal subcutaneous fat, were continuously perfused, and tissue dialysate concentrations of glycerol (lipolysis index), glucose, lactate, and pyruvate were determined. Comparisons were made with respective metabolite levels in venous plasma. After termination of intravenous insulin infusion, free insulin in plasma fell from 130 to 70 pM. At the same time, glucose levels in plasma and adipose tissue rose in parallel. However, the relative increase in glucose levels was greater in adipose tissue than in blood. On the other hand, the increase in glycerol concentration in adipose tissue (35%) was markedly less than that in venous plasma (250%). Lactate and pyruvate levels in adipose tissue and blood remained unchanged. After the resumption of intravenous insulin, free insulin in plasma rose to approximately 600 pM. At the same time, the glucose levels in blood and adipose tissue decreased rapidly, and the glycerol concentration in these tissues decreased to 50% of the baseline levels. The lactate and pyruvate levels in subcutaneous tissue increased briefly after insulin replacement, whereas the lactate but not pyruvate levels in blood showed a similar increase. The alpha- or beta-blocking agents phentolamine and propranolol in the ingoing tissue perfusate did not influence tissue glycerol at any time during the experiment. We concluded that insulin-induced changes in circulating metabolites only partly reflect variations in adipose tissue substrate kinetics. During insulin deprivation, glucose is accumulated in the adipose tissue extracellular compartment, probably because of reduced utilization by the adipocytes.(ABSTRACT TRUNCATED AT 250 WORDS)

Glucose determination in samples taken by microdialysis by peroxidase-catalyzed luminol chemiluminescence.

An automatic, luminometric assay of glucose in samples of the extracellular water space obtained by microdialysis is described. The assay involves oxidation by glucose oxidase (EC 1.1.3.4) and mutarotation of glucose by aldose mutarotase (EC 5.1.3.3.). The H2O2 formed is subsequently determined in a reaction catalyzed by horseradish peroxidase (EC 1.11.1.7) using luminol as electron donor. The assay is linear between 0.01 and 1 nmol in the cuvette. The detection limit, defined as 3 standard deviations of the reagent blank, was 0.008 mumol/liter in the cuvette. A complete oxidation of glucose is obtained within 4 min and 25 samples are automatically assayed within 75 min. Addition of microdialysate sample obtained from human adipose tissue in vivo did not interfere with the standard curves. Glucose added to microdialysate resulted in a complete recovery compared to a H2O2 standard. Analytical interference from different factors was investigated. No interference was observed up to the following concentrations: 5 mumol/liter epinephrine, 1 mumol/liter norepinephrine, 100 mumol/liter insulin, 500 mumol/liter pyruvate, 50 mmol/liter lactate, and 1 mumol/liter ascorbate. The glucose values with the present method correlated strongly (r = 0.984) with values obtained using a routine method involving glucose oxidase and peroxidase.

Free fatty acid determination by peroxidase catalysed luminol chemiluminescence.

A sensitive, specific, and partly automatic method for the analysis of free fatty acids is described. The assay involves activation of free fatty acids by acyl-CoA synthetase (EC 6.2.1.3) followed by oxidation of the thioesters by acyl-CoA oxidase. The H2O2 formed is determined in a reaction catalysed by horseradish peroxidase (EC 1.11.1.7) using luminol as electron donor. The assay has a linear range of 0.05 to 5 nmol of different free fatty acids (C10-C18) in the original sample. The efficiency of the method toward capric, lauric, myristic, palmitic, palmitoleic, stearic, oleic, and linoleic acid measured as recovery of light emission compared to that of H2O2 standards, was over 90%. AffiGel 501 was used to covalently bind the free thiol group in CoASH eliminating interference of this substance in the peroxidase-luminol reaction.

Perinatal opiate exposure. Effects on metabolism of xenobiotics and steroids in adult rat liver.

The effects of perinatal or neonatal morphine exposure on the hepatic steroid and xenobiotic metabolism in adult rats were studied. Early morphine exposure did not affect the 16 alpha-hydroxylation or 5 alpha-reduction of androstenedione in either sex, but decreased the 7 alpha- and 6 beta-hydroxylations in both sexes. Morphine exerted a suppressive effect on the ethoxyresorufin-O-deethylase activity and increased the ethoxycoumarin-O-deethylase activity in both sexes. Morphine exposure did not significantly affect its own N-demethylation or the total cytochrome P-450 content in the liver. Neonatal morphine exposure caused a significant decrease in body and testes weight in the adult male rat. We conclude that the effects of morphine are not confined to sex-differentiated pathways and are similar in both sexes.

Cytochrome P-450 b and c in the rat brain and pituitary gland.

A quantitative assessment of the levels of cytochromes P-450 b and P-450 c in the brains and pituitary glands of untreated and beta-naphthoflavone (BNF)-pretreated rats was made with polyclonal antibodies raised against hepatic P-450 b and c and the sensitive fluorometric assay of P-450 catalytic activity, namely, the O-deethylation of ethoxycoumarin (ETC). In the microsomal fraction of brains of untreated rats, the rate of formation of 7-hydroxycoumarin from ETC ranged between 0.1 and 20 pmol/min/mg of microsomal protein, which is approximately 0.01-2% of the level of hepatic microsomes of phenobarbital-induced rats. This brain activity was completely inhibited by anti P-450 b antibodies but was unaffected by anti P-450 c antibodies. As with hepatic P-450 b, metyrapone and chloramphenicol (100 microM) were good inhibitors of catalytic activity, whereas alpha-naphthoflavone (1 microM) was a poor inhibitor. No ETC O-deethylase activity was detectable in microsomes prepared from the pituitary glands of untreated rats. Upon pretreatment of rats with BNF, there was induction of ETC O-deethylase activity in the pituitary gland to a level of 3.3 +/- 1.5 pmol/min/mg of microsomal protein, but there was no significant increase in the level of activity in brain microsomes. Despite this, there was evidence of induction of P-450 c in both the brain and pituitary of BNF-pretreated rats since anti P-450 c antibodies inhibited brain activity by 55% and pituitary activity by 84%. The regional distribution of P-450 b and c in the hypothalamic-preoptic area and olfactory bulbs was examined. The level of ETC O-deethylase activity in the hypothalamic-preoptic area was not different from that in the whole brain, but in the olfactory bulbs activity was higher than that in whole brain, with a range of 0.1-52 pmol/min/mg of microsomal protein. The catalytic activity in the whole brain and in the olfactory bulbs was inhibited by anti P-450b but not by anti P-450c antibodies. Neither estradiol, testosterone, dehydrotestosterone, nor 5 alpha-androstane,3 beta,17 beta-diol (100 microM) competitively inhibited ETC O-deethylase activity, indicating that P-450 b is not responsible for the steroid hydroxylations previously reported in the brain. BNS pretreatment of rats did not cause a consistent increase in ETC O-deethylase upon BNF induction. However, there was an induction of P-450 c in the olfactory bulbs since catalytic activity was inhibited with anti P-450c antibodies.(ABSTRACT TRUNCATED AT 400 WORDS)

Activation of sexual behaviour in castrated rats: the role of oestradiol.

Sexual behaviour was induced in castrated male rats with oestradiol-17 beta- or testosterone-filled constant-release implants. Testosterone-induced sexual behaviour was unaffected by treatment with the 5 alpha-reductase inhibitor 17 beta-N,N-diethylcarbamoyl-4-aza-5 alpha-androstan-3-one (4-MA; 16.7 mg/day) but treatment with the aromatization inhibitor 1,4,6-androstatriene-3,17-dione (ATD; 10 mg/day) prevented testosterone from inducing the behaviour. Sexual behaviour could be activated in castrated rats treated with testosterone plus ATD by treatment with 4-MA or with implants filled with a low dose of oestradiol. Lordosis behaviour induced in ovariectomized rats with testosterone-filled implants and progesterone was blocked by ATD treatment and could not be activated with 4-MA but oestradiol implants restored the display of lordosis in the testosterone plus ATD-treated females. 4-MA inhibited the in-vitro formation of [14C]5 alpha-dihydrotestosterone from [14C]testosterone by combined preoptic and hypothalamic tissue at all doses tested and a high dose of oestradiol exerted a similar effect. The results suggest that androgen aromatization is required for testosterone-activated female sexual behaviour but not for testosterone-activated male sexual behaviour. It is suggested that oestradiol normally acts to control the sexual behaviour of male rats by modifying neural androgen metabolism.

Offspring of women with nonorganic psychosis: mother-infant interaction at one year of age.

Mother-infant interaction during feeding and in an unstructured play situation was studied in the home at 1 year of age in 46 index mother-infant pairs in which the mother had a history of nonorganic psychosis and in 80 demographically similar control pairs. As was true at the five previous observation ages, some aspects of the interaction were significantly more negative in index than control pairs. Index mothers showed increased tension and uncertainty regarding the infant's needs, increased physical contact during feeding and a discrepancy between the intonation and content of the mother's verbal contact with the infant. Index infants did not differ from controls in behavior in interaction. Across the entire first year, the Cycloid and Schizophrenic mothers deviated most frequently from controls, while the Affectives' interaction was more negative than controls' for the first time at the 1-year observation.

Offspring of women with nonorganic psychosis: mother-infant interaction at three-and-a-half and six months of age.

Mother-infant interaction during feeding and in an unstructured play situation was studied in the home at 3.5 and 6 months of age in index mother-infant pairs in which the mother had a history of nonorganic psychosis (n = 48 and 52 at 3.5 and 6 months, respectively) and in demographically similar control pairs (n = 80 and 79). Interaction was significantly more negative in index than control cases at both ages, index cases showing decreased maternal and infant social contact and reduced maternal sensitivity to the infant's needs. Schizophrenic and Cycloid groups evidenced more negative interaction characteristics than did their matched controls, while the Affective group was not generally more negative than its controls.

Offspring of women with nonorganic psychosis: mother-infant interaction at three and six weeks of age.

Mother-infant interaction during feeding and in an unstructured play situation was studied in the home at 3 weeks and 6 weeks of age in index mother-infant pairs in which the mother had a history of nonorganic psychosis (n = 42 and 51 at 3 and 6 weeks, respectively) and in demographically similar control pairs (n = 60 and 78). At both ages, interaction was significantly more negative in index than control cases, index mothers showing increased tension and a lack of harmony, decreased social contact, and reduced sensitivity to the infant's needs. Fewer significant differences were found between index and control infants. Mothers in the Schizophrenic, Cycloid and Nonendogenous groups evidenced more negative interaction characteristics than did their matched controls, but the Affective group was not in any way more negative than its controls.

Selective inhibition by chloramphenicol of cytochrome P-450 isozymes in rat lung and liver involved in the hydroxylation of n-hexane.

Treatment of rats with chloramphenicol causes a dose-dependent and regioselective inhibition of the metabolism of the organic solvent n-hexane in both liver and lung microsomes. A dose of chloramphenicol of 100 mg kg-1 administered i.v. or i.p. results in more than 50% inhibition of 2-hexanol formation catalyzed by microsomes from both organs, but causes no inhibition of 1-hexanol formation. The effects of chloramphenicol on 3-hexanol formation are somewhat organ-specific. In the liver 3-hexanol formation is inhibited to almost the same extent as 2-hexanol formation, whereas in the lung the inhibition of the formation of 3-hexanol is markedly less. Phenobarbital induces n-hexane metabolism in the liver but not the lung, but decreases the inhibitory potency of chloramphenicol toward both organs. In vitro chloramphenicol causes both reversible and irreversible inhibition of 2-hexanol formation in control lung microsomes. The irreversible inhibition is accompanied by the covalent binding of metabolites of chloramphenicol to the lung microsomes. The covalent binding is completely inhibited by antibodies to the major phenobarbital-induced isozyme of rat liver cytochrome P-450. These antibodies also cause more than 90% inhibition of 2-hexanol formation by lung microsomes. The results suggest that chloramphenicol acts as a selective suicide substrate of a constitutive isozyme of rat lung cytochrome P-450 involved in the 2-hydroxylation of n-hexane.

Offspring of women with nonorganic psychosis: mother-infant interaction at three days of age.

Mother-infant interaction during a feeding was studied at about 3 days of age in 51 index mother-infant pairs in which the mother had a history of nonorganic psychosis and in 73 demographically similar control pairs. Interaction was significantly more negative and deviant in index than control cases, index mothers establishing a significantly more negative emotional climate and less harmony in feeding, evidencing more tension and uncertainty, and showing less social contact toward the infant. Index infants showed significantly less social contact toward the mother and index pairs less reciprocal visual contact than did controls. While the Schizophrenic, Cycloid and Nonendogenous Psychoses groups each differed from their matched controls on a number of variables, the Affective group was not in any way more negative than its controls on interaction.

Offspring of women with nonorganic psychosis: fear of strangers during the first year of life.

Fear of strangers ( FOS ) during the infant's first year was studied in 46 "high-risk" offspring of index mothers with a history of nonorganic psychosis and in 80 demographically similar control offspring. FOS was measured in the home by a standardized test at 1 year of age and by repeated interviews with the mother during the first year. As compared with controls, the total index group, and the subgroups of offspring of mothers with Schizophrenia and Cycloid Psychosis, significantly more often showed a total absence of FOS in the test at 1 year, as well as during the entire first year. FOS at 1 year was unrelated both to serious active psychiatric disturbance in index mothers during the infant's first year of life, and the infant's sex, in index and control groups.

Offspring of women with nonorganic psychosis: infant attachment to the mother at one year of age.

Attachment to the mother at 1 year of age was studied in 46 "high-risk" offspring of index mothers with a history of nonorganic psychosis and in 80 demographically similar control offspring. Attachment was measured in the home in a standardized manner by a modified version of Ainsworth's Strange Situation procedure. Three different patterns, representing "secure attachment" (Type B), "anxiously avoidant attachment" (Type A) and "anxiously ambivalent attachment" (Type C), were defined. While no significant difference on attachment type was found between the total index versus control group, a significantly increased rate of anxious attachment (A + C) was found for offspring of schizophrenics but no other diagnostic group. Attachment type was unrelated to index mothers' psychiatric hospitalization, psychotic status and known mental disturbance during the infants' first year of life, as well as to the infants' sex.

Offspring of women with nonorganic psychoses. Development of a longitudinal study of children at high risk.

This article describes the development of a prospective, longitudinal study of 88 "high-risk" offspring of pregnant index women with a history of nonorganic psychoses and 104 offspring of demographically-similar pregnant control women. The maternal psychoses represented the diagnostic categories, Schizophrenia, Cycloid Psychosis, Affective Illness, Psychogenic Psychosis, Postpartum Psychosis and Other (remaining) Psychoses. The first phase of the study began during pregnancy and continued until the offspring reached 2 years of age. Selected characteristics of the mothers, the offspring and their environments were investigated during this project phase.

Suicide inactivation of rat liver cytochrome P-450 by chloramphenicol in vivo and in vitro.

Intraperitoneal administration of chloramphenicol (100 mg/kg) to phenobarbital-treated rats causes 50% inhibition of liver microsomal 7-ethoxycoumarin and 1,1,2,2 tetrachloroethane metabolism but has no effect on the level of cytochrome P-450 detectable as its carbon monoxide complex or on the NADPH-cytochrome c reductase (EC 1.6.2.4) activity. Both the endogenous NADPH oxidase activity and the enzymatic reduction of cytochrome P-450 are inhibited by chloramphenicol treatment, whereas the Km and Ks for ethoxycoumarin and the cumene hydroperoxide- or iodosobenzene-supported deethylation of ethoxycoumarin are unaffected, suggesting that impaired electron transport to cytochrome P-450 may be the cause of the loss of enzymatic activity. Administration of [14C]chloramphenicol (100 mg/kg) leads to the covalent binding of 0.7 nmole of metabolite(s) per nanomole of the major cytochrome P-450 isozyme. Alkaline hydrolysis of a cytochrome P-450 fraction obtained by chromatography of solubilized 14C-labeled microsomes on octylamino-Sepharose releases oxalic acid and chloramphenicol oxamic acid, whereas enzymatic digestion releases N-epsilon-chloramphenicol oxamyl lysine in addition. These data obtained with radiolabeled chloramphenicol suggest that the same metabolic pathways which lead to the inactivation of cytochrome P-450 in vitro are also operative in vivo.

Chain length heterogeneity of nucleosomal DNA in mouse liver after dimethylnitrosamine administration.

The effect of dimethylnitrosamine on the nucleosomal structure of mouse liver chromatin was studied. After a single oral dose of dimethylnitrosamine (2-75 mg/kg body weight 45 min before sacrifice) liver nuclei were isolated and incubated with micrococcus nuclease. Nucleosomes were separated on sucrose density gradients. There were no differences in nucleosomal sedimentation velocities between preparations from control and dimethylnitrosamine treated animals. The supernatant obtained after centrifugation of the lysed nuclei (2 min at 4,000 gav) and nucleosomal peak fractions were used for isolation of DNA. DNA was heat denatured in 7 M urea or formamide. After electrophoresis on polyacrylamide gels areas under mononucleosomal DNA and smaller fragments were measured and compared with the total DNA area. The increase in DNA fragmentation was dimethylnitrosamine dose response dependent. When expressed as per cent of controls it amounted to 106% for 2 mg; 115% for 10 mg; 127% for 25 mg; 164% for 75 mg dimethylnitrosamine/kg body weight. A good correlation between mobility and log of chain length of phi chi 174 RF DNA-Hae III digest was obtained in nondenaturing 5% polyacrylamide gels and denaturing non-aqueous formamide polyacrylamide gels but not in 12% polyacrylamide gels containing 7 M urea. DNA of mononucleosomal peak fractions contained 200 and that of dinucleosomal peak fractions 400 nucleotides. Fragmentation of DNA was closely related to in vivo dimethylnitrosamine treatment but was not detected in measurements of protein-DNA complexes in the chromatin. It was disclosed on denaturation of DNA followed by polyacrylamide gel electrophoresis.